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  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/02—Antineoplastic agents specific for leukemia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/7056—Lectin superfamily, e.g. CD23, CD72
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

• a mutation may result in a substitution that is non-conservative. Such mutations are acceptable to the extent that the chimeric polypeptide is capable of binding to an NKG2D ligand. More detailed descriptions of different types of NKG2D-Fc chimeras are provided below.
• the NKG2D portion of the NKG2D-Fc chimera used for methods provided in this disclosure may be a full length NKG2D polypeptide.
• the full length sequence of NKG2D has been described in the literature. See, for example, Accession: NP_031386.
• alternative splice variants of NKG2D have been described. For purposes of the instant invention, any one of such alternatively spliced variants may be used, provided that the resulting polypeptide, when constructed as an NKG2D-Fc chimera, is capable of binding its ligans(s).
• the NKG2D-Fc construct may contain a fragment of the extracellular domain, wherein the C-terminus of the domain ends at amino acid residue 231 , 230, 229, 228, 227, 226, 225, 224, 223, 222, 221 , 220, 219, 218, 217, 216, 215, 214, 213, 212, 21 1 , 210, 209, 208, 207, 206, 205, and so forth. Such deletions at each end of the extracellular domain of the NKG2D sequence may be combined.
• NKG2D-Fc chimera
• the NKG2D fragment corresponds to amino acid residues 92 to 232 of the murine NKG2D.
• the same approach may be employed for NKG2D sequences derived from any other species that are known to develop cancer.
• the NKG2D fragment of NKG2D-Fc may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid changes, such as deletions, insertions and substitutions, as long as the NKG2D-Fc retains its ligand binding activity.
• certain polymorphic allotypes of a given isotype may influence affinity for an Fc receptor. Indeed, there are allelic variants of activating FcRs that will significantly affect the affinity for certain antibody isotypes. For example, the Fc ⁇ RIIIa receptor 158V allotype displays a higher affinity for human immunoglobulin Gi and increased antibody-dependent cellular cytotoxicity (Cartron G. et al., 2002, Blood, 99: 754-758).
• the NKG2D-Fc chimera useful for the methods provided in this disclosure contains an Fc portion.
• the structure of Fc fragments generally is known in the art. Briefly, the Fc region of a typical IgG molecule is a symmetric homodimer of the carboxy-terminal portion of heavy chains and is composed of the C H 2 and C H 3 domains, which are separated from the Fab by a flexible hinge region. The Fc region is stabilized by non-covalent interactions between domains. The Fc region interacts with FcRs to exert effector functions or to regulate the catabolism of IgG.
• the NKG2D chimera contains an N-terminal modified IL-2 signal sequence, which allows for optimal expression and secretion of NKG2D-Fc construct. See, for example, Zhang et al., 2004, J. Gene Med., 7:354-65.
• the NKG2D chimera contains a signal peptide derived from the polypeptide sequence of CD33.
• the CD33 signal peptide may correspond to amino acid residues 1 -16 of the CD33 polypeptide sequence.
• One of ordinary skill in the art will understand that there are a number of other suitable signal peptide sequences that may be used to practice the methods provided in this disclosure.
• a region of the NKG2D portion of the NKG2D-Fc chimera may also contribute to dimerization.
• NKG2D-Fc chimera is produced by conventional recombinatory DNA methods.
• an NKG2D-Fc chimera is produced as a single (e,g., contiguous) recombinant polypeptide.
• two or more portions of NKG2D-Fc are produced as separate fragments and are subsequently linked together to yield an NKG2D-Fc molecule.
• NKG2D-Fc can be purified by any suitable means such as
• the NKG2D-Fc chimeric molecules bind ligands with high affinity of 10 "4 or less, 10 "7 M or less, or with subnanomolar affinity, e.g., 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1 nM or even less.
• the binding affinity of the 30 NKG2D-Fc molecule for its ligands is at least 5 x 10 6 Ka, at least 1 x 10 7 Ka, at least 2 x 10 7 Ka, at least 1 x 10 8 Ka, or greater.
• NKG2D ligands Normally, expression of the NKG2D ligands appears to be confined to the gastrointestinal epithelium. Little expression is observed in quiescent epithelial cells, but higher levels of expression occur in rapidly proliferating cells. Expression of the NKG2D ligands is also up-regulated in various transformed cells, particularly those of epithelial origin. Accordingly, provided herein are methods for treating cancer or symptoms of cancer in a subject. The methods comprise administering to the subject a therapeutically effective amount of NKG2D-Fc that binds NKG2D ligands in vivo.
• a "subject" as used herein is a mammalian subject having a disease, or at risk of developing a disease associated with an abnormal expression of at least one NKG2D ligand, such as cancer.
• the subject is a human subject having a cancer presenting elevated levels of one or more NKG2D ligands.
• the NKG2D ligands include MICA.
• NKG2D-Fc chimeras can function through the two major components of the immune system: the innate immunity and adaptive immunity.
• innate immunity or the innate immune system refers to non-specific host defense mechanisms against foreign pathogens.
• Innate immunity includes both physical barriers (e.g., skin, gastric acid, mucus or tears, as well as cells and active mechanisms such as NK cells, phagocytes and the complement system.
• NK cells represent a major component of the innate immune system. NK cells are cytotoxic, e.g., are able to attack cells that have been infected by microbes, as well as some kinds of tumor cells.
• NKG2D-Fc fusion protein can: (1) trigger complement dependent lysis (complement-dependent cytotoxicity or CDC); (2) trigger antibody dependent cellular cytotoxicity (ADCC); (3) promote the opsonization of myeloma cells for cross-presentation by dendritic cells; and, (4) antagonize the immunosuppressive effects of shed ligands.
• complement-dependent cytotoxicity or CDC complement-dependent cytotoxicity
• ADCC antibody dependent cellular cytotoxicity
• B 16-MICA B 16-MICA
• Figure 7 provides results from in vitro complement lysis in a murine lung tumor model.
• MDAC8 cells were employed. MDAC8 cells were derived from a primary lung tumor in triple knockout mice that are lacking GM-CSF, IFN- ⁇ and IL-3 (GM-CSF-/-, IFN- ⁇ -/- & IL-3-/-). The MDAC8 cells were incubated with rabbit complement (1 :20 dilution) and either NKG2D-Fc as described in the previous section of the Example, or control IgG2a.
• NKG2D-Fc chimera caused lysis of the target MDAC8 cells in a dose-dependent fashion, while IgD2a had no effect.
• the result provides in vivo evidence that NKG2D-Fc can effectively induce complement-dependent cytotoxicity in the cancer model.
• an NKG2D-Fc chimera may be examined in vivo using standard methods available to those skilled in the art. For example, varying numbers of plamacytoma cells may be injected subcutaneously into syngeneic adult Balb/c mice, followed by systematic administration of varying doses of the NKG2D-Fc fusion proteins or IgG2a isotype controls. Initially, an optimal fusion protein dose and schedule for inhibiting myeloma growth in the skin may be determined in a mouse model, starting typically with ⁇ 5 animals. In these studies, tumor growth may be monitored at 2-3 day intervals, the product of tumor diameters determined, and mice sacrificed when tumors reach 2 cm in greatest diameter or ulcerate. Once an active regimen has been established, experimental groups will be increased, for example to 10-20, so as to achieve statistical significance.
• Anti-CTLA-4 antibodies can then be purified from supernatants of the 9H10 hybridoma using a HiTrapTM protein A sepharose column (Amersham Bioscience), as earlier reported (Enzler et al., 2007). Subsequently, conditions may be established, in which combinations of GM-CSF secreting plamacytoma vaccines and CTLA-4 blockade mediate anti-tumor activity against pre-existing plasmacytomas.
• a typical administration regime may involve three daily injections of 100 ⁇ g of anti-CTLA-4 mAbs with vaccination (IxIO 6 irradiated cells), whereas initial tumor challenges may consist of 5xlO 5 cells.
• Vaccination is initiated on day zero, and depending on the efficiency of tumor rejection, progressively delay the onset of therapy to define conditions in which the combination treatment shows only modest activity. It is then determined whether the addition of the NKG2D-Fc fusion protein to the GM-CSF secreting vaccine/CTLA-4 blockade combination under these conditions enhances myeloma cell destruction. Detailed immune analysis can be undertaken as described above if therapeutic potency is increased (comparisons with the GM-CSF secreting vaccine/CTLA-4 combination alone would also be made). In the absence of enhanced protection, tumor infiltrates may be initially characterized and then additional analysis are performed, depending on which cell types are most impacted, as discussed above.
• NKG2D-Fc fusion protein binds soluble NKG2D ligands in an ELISA
• sera collected longitudinally from XBP-I transgenic mice may be evaluated for the presence of shed ligands.
• correlation between the production of one or more of the identified NKG2D ligands and upregulation of ERp5 expression in transgenic plasma cells may be established. This may be determined by employing flow cytometry using the commercially available anti-human ERp5 sera, which cross-reacts with the murine protein.
• the potential immunosuppressive effects of shed murine NKG2D ligands may initially be evaluated using techniques similar to those used for sMICA in clinical samples.
• NKG2D pathway as a broad target for cancer immunotherapy in a wide variety of cancer types that present abnormal levels of NKG2D ligand expression.
• fully human anti-MICA monoclonal antibodies may become available for clinical use.
• the methods described herein employing NKG2D-Fc provide added advantage of eliciting broader effects in cancer therapy by virtue of its ability to bind multiple types of ligands.

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• 2009
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